Methods and compositions for the detection of bacterial endotoxins

ABSTRACT

The invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1→3) β-D-glucan.

FIELD OF THE INVENTION

[0001] This invention relates generally to an amebocyte lysatepreparation for use in the detection and/or quantification of abacterial endotoxin in a sample, and more particularly to anendotoxin-specific amebocyte lysate preparation having reduced Factor Gactivity for use in the detection and/or quantification of a bacterialendotoxin in a sample.

BACKGROUND OF THE INVENTION

[0002] Bacterial endotoxins, also known as pyrogens, are thefever-producing byproducts of Gram negative bacteria and can bedangerous or even deadly to humans. Symptoms of infection may range fromfever, in mild cases, to death. In order to promptly initiate propermedical treatment, it is important to identify, as early as possible,the presence of an endotoxin and, if possible, the concentration of theendotoxin in the subject of interest. Similarly, the U.S. Food and DrugAdministration (USFDA) requires certain manufacturers to establish thattheir products, for example, parenteral drugs and medical devices, arefree of detectable levels of Gram negative bacterial endotoxin.

[0003] To this end, a variety of methods have been developed for use inthe detection of bacterial endotoxins. A currently preferred methodinvolves the use of amebocyte lysate (AL) produced from the hemolymph ofa horseshoe crab, for example, a horseshoe crab selected from the groupconsisting of Limulus polyphemus, Tachpleus gigas, Tachypleustridentatus, and Carcinoscorpius rotundicauda. Amebocyte lysatesproduced from Limulus, Tachpleus, and Carcinoscorpius maybe referred toas LAL, TAL, and CAL, respectively.

[0004] Presently, LAL is employed in bacterial endotoxin assays ofchoice because of its sensitivity, specificity and relative ease foravoiding interference by other components that may be present in asample of interest. LAL, when combined with a sample containingbacterial endotoxin, reacts with the endotoxin to produce a product, forexample, a gel or chromogenic product, that can be detected, forexample, either visually or by the use of an optical detector.

[0005] The endotoxin-mediated activation of LAL is well understood andhas been thoroughly documented in the art. See, for example, Levin etal. (1968) Thromb. Diath. Haemorrh. 19: 186, Nakamura et al. (1986) Eur.J. Biochem. 154: 511, Muta et al. (1987) J. Biochem. 101: 1321, and Hoet al. (1993) Biochem. & Mol. Biol. Int. 29: 687. When bacterialendotoxin is contacted with LAL, the endotoxin initiates a series ofenzymatic reactions, referred to in the art as the Factor C pathway,that involve at least three serine protease zymogens called Factor C,Factor B and pro-clotting enzyme (see FIG. 1). Briefly, upon exposure toendotoxin, the endotoxin-sensitive factor, Factor C is activated.Activated Factor C thereafter hydrolyses and activates Factor B,whereupon activated Factor B activates proclotting enzyme to produceclotting enzyme. The clotting enzyme thereafter hydrolyzes specificsites, for example, Arg¹⁸-Thr¹⁹ and Arg⁴⁶-Gly⁴⁷ of coagulogen, aninvertebrate, fibrinogen-like clottable protein, to produce a coagulingel. See, for example, U.S. Pat. No. 5,605,806.

[0006] Although the clotting cascade of LAL initially was consideredspecific for endotoxin, it was later discovered that (1→3)-B-D glucansalso activate the clotting cascade of LAL through a unique enzymaticpathway, referred to in the art as the Factor G pathway (see FIG. 1).Upon exposure to (1→3)-B-D glucan, Factor G is activated to produceactivated Factor G. Activated Factor G thereafter converts theproclotting enzyme into clotting enzyme, whereupon the clotting enzymeconverts coagulogen into coagulin, similar to the case with endotoxin.Accordingly, the coagulation system of LAL, like the mammalian bloodcoagulation system, consists of at least two coagulation cascades whichinclude an endotoxin-mediated pathway (the Factor C pathway), and a(1→3)-B-D glucan-mediated pathway (the Factor G pathway). See, forexample, Morita et al. (1981) FEBS Lett. 129: 318-321 and Iwanaga et al.(1986) J. Protein Chem. 5: 255-268.

[0007] In view of the Factor C and Factor G pathways of LAL, thedetection of bacterial endotoxin in a sample can, under certaincircumstances, become ambiguous. As a result, attempts have been made toincrease the specificity of LAL for endotoxin, i.e., to produce anendotoxin-specific amebocyte lysate preparation.

[0008] In one approach, polysaccharide based Factor G inhibitors arecombined with amebocyte lysate to reduce or eliminate clotting inducedby (1→3)-B-D glucan present in the biological sample, i.e., inhibit theFactor G cascade. See, for example, U.S. Pat. Nos.: 5,155,032;5,179,006; 5,318,893; 5,474,984; and 5,641,643.

[0009] In an alternative approach, several groups have attempted toremove Factor G from LAL thereby to produce a Factor G depletedamebocyte lysate that is insensitive to (1→3)-B-D glucan. For example,Obayashi et al. (1985) Clin. Chim. Acta 149:55-65 disclose a method forfractionating coagulation enzymes in LAL and then recombining only thosefactors involved in the endotoxin induced coagulation cascade (i.e., theFactor C cascade) to produce a Factor G depleted amebocyte lysate. Theresulting lysate, however, may not only lack Factor G but also othercomponents required for a complete Factor C cascade. The reconstitutedlysate produced by this procedure, apparently does not produce a naturalcoagulin type clot and can be used only with synthetic chromogenicsubstrates.

[0010] U.S. Pat. No. 5,401,647 discloses a method for removing Factor Gfrom LAL by combining LAL with (1→3)-B-D glucan immobilized on aninsoluble carrier. Once bound to the carrier via the (1→3)-B-D glucanmoiety, the Factor G can thereafter be removed from the LAL to produce aFactor G depleted lysate. Similarly, U.S. Pat. No. 5,605,806 disclosesan immunoaffinity based method using a Factor G specific antibody toremove Factor G from LAL thereby to produce a Factor G depletedamebocyte lysate.

[0011] There still exists, however, a demand for an endotoxin-specificamebocyte lysate that can be produced economically in commercialquantities. A method for producing such an amebocyte lysate should berapid, reproducible, inexpensive, simple to conduct, and preferablyshould result in an amebocyte lysate that can be used in a reliable, andquantitative determination of endotoxin in a sample of interest.

SUMMARY OF THE INVENTION

[0012] The invention features improved amebocyte lysate preparationshaving reduced Factor G activity, methods of making such lysatepreparations, and methods of using such lysate preparations in thedetection and/or quantitation of one or more bacterial endotoxins in asample of interest.

[0013] In one aspect, the invention provides a method of producing anendotoxin-specific amebocyte lysate preparation for use in the detectionof bacterial endotoxins in a sample. The amebocyte lysate preparation isrendered endotoxin-specific by the reduction and/or elimination ofFactor G activity in the preparation. The amebocyte lysate preparationof the invention is produced by (a) admixing crude amebocyte lysate,i.e., amebocyte lysate reactive with both endotoxin and (1→3)-B-Dglucan, with a surfactant in an amount sufficient to produce a solutioncontaining a precipitate; and (b) separating the precipitate from thesolution thereby to produce an amebocyte lysate preparation which isless reactive with a (1→3)-β-D glucan than is the crude amebocytelysate. The precipitate produced by addition of surfactant to crudelysate may contain any component necessary for a complete Factor Gcascade, however, the production of a precipitate actually containingFactor G is preferred.

[0014] The amebocyte lysate preparation produced by the methodologiesdescribed herein comprises all the components necessary for a completeFactor C cascade, i.e., is still capable of producing a coagulin gel viathe endotoxin-mediated pathway. Accordingly, the resulting amebocytelysate preparation is capable of reacting with a bacterial endotoxin,e.g., a bacterial endotoxin produced by Gram negative bacteria, toproduce a coagulin clot.

[0015] It is contemplated that any surfactant (otherwise known as asurface active agent or detergent) which produces a precipitate whenadded to crude amebocyte lysate, wherein the precipitate once removedfrom the lysate results in a reduction of Factor G activity, may be usedin the practice of the invention. The surfactant, however, preferably isa zwitterionic surfactant, i.e., a surfactant having a headgroupcontaining both a negatively charged chemical moiety and a positivelycharged chemical moiety. Examples of zwitterionic surfactants includebetaines and sulfobetaines, however, sulfobetaine-type surfactants arepreferred. Preferred sulfobetaine-type surfactants include, withoutlimitation, n-octyl- N, N-dimethyl-3-ammonio-1-propanesulfonate;n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. The sulfobetaine-typesurfactant n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate,however, is most preferred.

[0016] In one embodiment, the method comprises the additional step ofremoving from or otherwise reducing the concentration of the addedsurfactant in the solution. The surfactant may be removed, for example,by chromatographic separation using, for example, a suitable ionexchange resin or, alternatively, by any other means known in the artfor removing a particular surfactant from an aqueous solution. In apreferred method, the surfactant is removed by conventional organicsolvent extraction. Any organic solvent that dissolves the surfactant ofinterest and is compatible with amebocyte lysate may be used in thesolvent extraction step, however, for the reasons discussed below,chloroform is preferred.

[0017] In another embodiment, the sensitivity to bacterial endotoxin ofan amebocyte lysate preparation having reduced Factor G activity can beenhanced by the addition of exogenous (1→3)-β-D glucan to the amebocytelysate preparation. In particular, (1→3)-β-D glucan is added to thelysate preparation in an amount sufficient to enhance the sensitivity ofthe lysate preparation to endotoxin relative to a similar amebocytelysate preparation without exogenously added (1→3)-β-D glucan. Withoutwishing to be bound by theory, it appears that exogenously added(1→3)-β-D glucan acts synergistically with the endotoxin mediatedpathway. It is understood, however, that the same amount of (1→3)-β-Dglucan when added to crude amebocyte lysate, i.e., amebocyte lysate thatis reactive with both endotoxin and (1→3)-β-D glucan, likely wouldinduce the production of a coagulin gel via the Factor G cascade. Ineffect, during the practice of this particular embodiment of theinvention, a substrate or initiator of the Factor G cascade is added tothe amebocyte lysate preparation of the invention.

[0018] Although it is contemplated that any amount of (1→3)-β-D glucanthat enhances the sensitivity of the amebocyte lysate to the endotoxinrelative to similar amebocyte lysate without the exogenously added(1→3)-β-D glucan may be used in the practice of the invention, theoptimal amount of exogenous (1→3)-β-D glucan for enhancing theendotoxin-specific cascade in a particular lysate can be determined byroutine experimentation. For example, the optimal concentration can bedetermined by adding different amounts of a particular (1→3)-β-D glucanto crude amebocyte lysate, i.e., amebocyte lysate reactive with bothendotoxin and (1→3)-β-D glucan. The optimal amount of the (1→3)-β-Dglucan to be added to the amebocyte lysate preparation of the invention,can be determined using, for example, a kinetic turbidimetric assaywhereby the optimal amount is the amount of (1→3)-β-D glucan thatinduces the fastest coagulin clot formation in crude amebocyte lysate.This assay protocol is exemplary, and it is understood that the skilledartisan may use a variety of other assays, for example, a gel-clotassay, an end-point turbidimetric assay, or a chromogenic assay, todetermine the optimal amount of (1→3)-β-D glucan to be added to thelysate of interest.

[0019] It is contemplated that any (1→3)-β-D glucan that induces theFactor G cascade in crude amebocyte lysate can be used to enhance thesensitivity of the endotoxin mediated pathway in the amebocyte lysatepreparation of the invention. Preferred (1→3)-β-D glucans include,without limitation, cotton extract; rinses from cellulose acetatemembranes; curdlan; pachyman; scleratan; leutinan; schizophyllan;coriolan; laminaran; and laminarin. Laminarin, however, currently ismost preferred.

[0020] In another aspect, the invention provides an amebocyte lysatepreparation having reduced Factor G activity, i.e., an amebocyte lysatehaving reduced reactivity to (1→3)-β-D glucans relative to crude lysateproduced by the aforementioned methodologies. In one embodiment, such acomposition may comprise (i) an amebocyte lysate preparation havingreduced Factor G activity or, most preferably, an amebocyte lysatepreparation depleted of Factor G activity, and (ii) exogenously added(1→3)-β-D glucan, wherein the (1→3)-β-D glucan is added in an amountsufficient to enhance the sensitivity of the amebocyte lysatepreparation to endotoxin relative to a similar amebocyte lysatepreparation without the exogenously added (1→3)-β-D glucan.Determination of the optimal amount of a particular (1→3)-β-D glucan forenhancing sensitivity of the lysate to endotoxin has been discussedpreviously.

[0021] In another aspect, the invention provides methods for detectingand/or quantitating the amount of a bacterial endotoxin in a sample. Theimprovement in such methods resides in the use of the amebocyte lysatepreparation of the invention.

[0022] The foregoing and other objects, features and advantages of thepresent invention will be made more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The objects and features of the invention may be betterunderstood by reference to the drawings described below in which,

[0024]FIG. 1 is a schematic representation of the blood coagulationsystem of horseshoe crab amebocytes.

[0025]FIG. 2 is a flow chart showing an exemplary protocol for producingan amebocyte lysate preparation having reduced Factor G activity.

[0026]FIG. 3 is a densitometric representation of a Coomassie Bluestained sodium dodecyl sulfate (SDS) polyacrylamide gel in which lane 1contains molecular weight markers, lane 2 contains crude Limulusamebocyte lysate, lane 3 contains precipitate removed from Limulusamebocyte lysate following the addition of a precipitating amountsurfactant, and lane 4 contains amebocyte lysate supernatant producedafter removal of the surfactant induced precipitate.

[0027]FIG. 4 is a graph showing the reactivity with laminarin of crudeLimulus amebocyte lysate and Limulus amebocyte lysate of the invention.Diamonds represent batch K2222L, squares represent batch L4941LB,triangles represent batch L1081LB, and circles represent batch L1711LB.

[0028]FIG. 5 is a graph showing the reactivity with a cellulose acetaterinse of crude Limulus amebocyte lysate and Limulus amebocyte lysate ofthe invention. Diamonds represent batch K2222L, squares represent batchL4941LB, triangles represent batch L1081LB, and circles represent batchL1711LB.

[0029]FIG. 6 is a graph showing the reactivity with cotton extract ofcrude Limulus amebocyte lysate and Limulus amebocyte lysate of theinvention. Diamonds represent batch K2222L, squares represent batchL4941LB, triangles represent batch L1081LB, and circles represent batchL1711LB.

[0030]FIG. 7 is a graph showing the sensitivity to endotoxin of Limulusamebocyte lysate of the invention in the presence and absence ofexogenously added (1→3)-β-D glucan. The circles-represent lysate withoutexogenously added laminarin and the boxes represent lysate withexogenously added laminarin.

DETAILED DESCRIPTION OF THE INVENTION

[0031] As will be more fully described below, this invention is based,in part, upon the discovery of an inexpensive and reliable method forproducing an endotoxin-specific amebocyte lysate preparation. Theresulting amebocyte lysate preparations are useful in the detectionand/or quantitation of a bacterial endotoxin in a sample of interest.

[0032] In particular, the method is based upon a protocol for reducingor preferably depleting amebocyte lysate of Factor G activity, with theresulting lysate being less reactive to (1→3)-β-D glucan than untreatedamebocyte lysate. In addition, the invention is based, in part, upon thediscovery that (1→3)-β-D glucan, when exogenously added to an amebocytelysate preparation depleted of Factor G activity (for example, by theremoval of Factor G, or by the addition of Factor G inhibitors, forexample, Factor G inhibitors of the type described in U.S. Pat. Nos.5,155,032; 5,179,006; 5,318,893; 5,474,984; and 5,641,643, thedisclosures of which are incorporated herein by reference) can enhancethe sensitivity of the resulting amebocyte lysate preparation toendotoxin. The invention, therefore, provides an endotoxin-specificamebocyte lysate preparation for use in reliably detecting and/orquantitating a bacterial endotoxin in a sample of interest.

[0033] The amebocyte lysate preparation of the invention is produced by:(a) admixing a sample of crude amebocyte lysate, i.e., amebocyte lysatethat is reactive with both endotoxin and (1→3)-β-D glucan, with asurfactant in an amount sufficient to produce a solution containing aprecipitate; and (b) separating the precipitate from the solutionthereby to produce an amebocyte lysate preparation which is lessreactive with a (1→3)-β-D glucan than the crude amebocyte lysate. Theresulting amebocyte lysate preparation preferably still comprises allcomponents necessary for the Factor C cascade and, therefore, is capableof producing a coagulin gel following the addition of endotoxin.

[0034] As used herein, the term, “amebocyte lysate” is understood tomean any lysate produced by the lysis of blood cells (amebocytes)extracted from the hemolymph of a horseshoe crab. Preferred horseshoecrabs include crabs belonging to the Limulus genus, for example, Limuluspolyphemus, the Tachpleus genus, for example, Tachpleus gigas, andTachypleus tridentatus, and the Carcinoscorpius genus, for example,Carcinoscorpius rotundicauda. As used herein, the term, “crude amebocytelysate” is understood to mean any amebocyte lysate that is capable ofproducing a coagulin clot in the presence of an endotoxin, for example,an endotoxin produced by Gram negative bacteria, and a (1→3)-β-D glucan,for example, laminarin.

[0035] As used herein, the term, “Factor G” is understood to mean anyprotein or polypeptide that acts as a serine protease zymogen and iscapable of initiating the production of a coagulin gel-clot in crudeamebocyte lysate following exposure to (1→3)-β-D glucan. The isolationand characterization of horseshoe crab Factor G has been discussedextensively in the art (see, for example, Seki et al. (1994) J. Biol.Chem. 269: 1370-1374, the disclosure of which is incorporated herein byreference) and, therefore, is not discussed in detail herein.

[0036] As used herein, the term, “(1→3)-β-D glucan” is understood tomean any water soluble polysaccharide, disaccharide or derivativethereof that is (i) capable of inducing formation of a coagulin clot incrude Limulus amebocyte lysate, and (ii) contains at least two β-Dglucosides, as defined in formula I below, connected by a (1→3)-β-Dglycosidic linkage. It is contemplated that such a polysaccharide orderivative thereof, in addition to containing a (1→3)-β-D glycosidiclinkage may also contain glucoside moieties connected by a variety ofother glycosidic linkages, for example, via a (1→4)-β-D glycosidiclinkage and/or by a (1→6)-β-D glycosidic linkage. It is contemplatedthat such (1→3)-β-D glucans may be isolated from a variety of sourcesincluding, without limitation, plants, bacteria, yeast, algae, andfungi, or alternatively may be synthesized using conventional sugarchemistries.

[0037] As used herein, the term “reactive with (1→3)-β-D glucan” refersto an amebocyte lysate, which in the presence of a (1→3)-β-D glucan iscapable of producing a product that can be detected in a conventionalgel-clot assay, end point-turbidimetric assay, kinetic turbidimetricassay or a chromogenic assay. Similarly, as used herein, the term“reactive with a bacterial endotoxin” refers to an amebocyte lysate,which in the presence of an endotoxin produced by a Gram negativebacteria is capable of producing a product that can be detected in aconventional gel-clot assay, end point-turbidimetric assay, kineticturbidimetric assay or a chromogenic assay.

[0038] As used herein, the term, “surfactant” is understood to mean anysurface active agent or detergent that is capable of producing aprecipitate when admixed with crude amebocyte lysate, wherein theprecipitate once removed from the lysate results in a reduction ofFactor G activity. As used herein, the term, “zwitterionic surfactant”is understood to mean any surfactant having a headgroup containing botha negatively charged chemical moiety and a positively charged chemicalmoiety. Examples of useful zwitterionic surfactants useful in thepractice of the instant invention include, without limitation, betaines(see, for example, formula II below) and sulfobetaines (see, forexample, formula III below), however, sulfobetaines currently are themost preferred.

[0039] wherein n can be 7, 9, 11, 13 or 15.

[0040] As used herein, the term, “precipitate” is understood to mean anyinsoluble material produced following admixture of crude amebocytelysate with a surfactant. It is contemplated that the precipitate maycontain Factor G and/or other, heretofore undiscovered, componentsnecessary for a functional Factor G cascade.

Preparation of Amebocyte Lysate Having Reduced Factor G Activity

[0041] A flow chart showing an exemplary protocol for producing anamebocyte lysate preparation having reduced Factor G activity, morepreferably, an amebocyte lysate preparation depleted of Factor Gactivity is shown in FIG. 1. It is contemplated that any crude amebocytelysate may be used as a starting material in the protocol shown in FIG.1.

[0042] Crude lysates may be produced using the procedure as originallydescribed in Levin et al. (1968) Thromb. Diath. Haemorrh. 19:186, withmodification. Briefly, blood (hemolymph) is harvested from horseshoecrab in a saline solution isotonic with sea water (about 3% NaCl (w/v))containing an anti-coagulant, for example, N-ethylmaleimide, caffeine,or Tween® 20. The amebocytes are washed with the same solution to removehemolymph factors and lysed by osmotic shock via exposure topyrogen-free water. After 24 hours, the amebocyte lysate is separatedfrom cellular debris by centrifugation. The preparation of crude lysatealso is discussed, for example, in Richard B. Prior, Ed., “ClinicalApplications of the Limulus Amebocyte Lysate Test” CRC Press, pp. 28-36and pp. 159-166, and in U.S. Pat. No. 4,322,217, the disclosures ofwhich are incorporated herein by reference.

[0043] In order to produce amebocyte lysate having reduced Factor Gactivity, surfactant is added to crude amebocyte lysate in an amountsufficient to produce a precipitate. As mentioned previously, it iscontemplated that any surfactant or detergent which produces aprecipitate upon addition to crude amebocyte lysate, wherein theprecipitate once removed from the lysate results in a reduction ofFactor G activity, may be used in the practice of the invention.Preferred surfactants include zwitterionic surfactants, i.e.,surfactants having a headgroup containing both a negatively chargedchemical moiety and a positively charged chemical moiety. Examples ofzwitterionic surfactants useful in the practice of the invention includebetaines and sulfobetaines, however, sulfobetaine-type surfactants arethe more preferred.

[0044] A family of sulfobetaine type detergents are availablecommercially from Calbiochem®, San Diego, Calif. under the tradenameZwittergent®. Sulfobetaine type detergents apparently retainzwitterionic character over a wide pH range. Currently preferredsulfobetaine-type surfactants include, without limitation, n-octyl- N,N-dimethyl-3-ammonio-1-propanesulfonate (also known as Zwittergent®3-08); n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (also known asZwittergent® 3-10); n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate(also known as Zwittergent® 3-12); n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (also known as Zwittergent®3-14); and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (alsoknown as Zwittergent® 3-16). The sulfobetaine-type surfactant isn-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent®3-14), however, is most preferred.

[0045] The optimal concentration of surfactant necessary to produce aprecipitate when combined with crude amebocyte lysate can be determinedby routine experimentation. For example, the skilled artisan may simplyadd increasing concentrations of a particular surfactant to crudeamebocyte lysate until a precipitate is produced. The amebocyte lysatepreparation following removal of the precipitate can then be tested forreduced Factor G activity using any of the conventional assays, forexample, gel-clot, end-point turbidimetric, kinetic turbidimetric, orchromogenic assays, well known and thoroughly document in the art. Withregard to the sulfobetaine type-detergents, preferred detergentconcentrations range from about 0.01% to about 0.6% (w/v) and mostpreferably from about 0.05% to about 0.25% (w/v). Specifically, withregard to the sulfobetaine-type surfactant n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent® 3-14), thisdetergent is preferably added to the crude lysate to a finalconcentration of from about 0.05% (w/v) to about 0.20% (w/v), mostpreferably about 0.12% (w/v) to produce a precipitate.

[0046] Optimal precipitation conditions may be determined by varying oneor more of detergent concentration, temperature, and time of incubation.For example, preferred incubation conditions include incubation attemperatures below about 20° C., most preferably about 2-15° C. forabout 2-24 hours. These conditions appear to preserve the activity ofthe lysate preparation during the precipitation step. For example, inthe case of Zwittergent® 3-14, the resulting mixture preferably isincubated at 2-8° C. for 8-24 hours. Following incubation, the resultingprecipitate can be removed by any conventional technique known in theart, for example, centrifugation followed by the removal of supernatantfrom a pellet of precipitate.

[0047] In a preferred embodiment, the concentration of the surfactantremaining in the supernatant, if necessary, is reduced to a level thatpermits the Factor C cascade to be operative. The extent of surfactantremoval to produce a lysate containing an operative Factor C cascade maybe determined by routine experimentation. For example, the amount ofsurfactant remaining in the supernatant can be determined, for example,by standard thin layer chromatography (TLC). Under certain circumstancesit may be preferable to remove at least about 35% of the surfactant fromthe supernatant, more preferably at least about 70%, and most preferablyat least about 90%. For example, consistent removal of at least about90% of the surfactant from the lysate, enables one to produceformulations of amebocyte lysate preparations with relatively definedcomponents. As a result, it may be easier to produce batches ofamebocyte lysate preparations having the same or similar activities. Itis understood, however, that it is possible to control the sensitivityof the resulting lysate preparation to endotoxin by altering theconcentration of residual detergent in the lysate preparation. Forexample, the more detergent removed, the more sensitive the lysate toendotoxin. Accordingly, in order to prepare a lysate having a desiredsensitivity to endotoxin, the amount of detergent removed from thelysate preparation may be altered by varying the detergent extractionconditions discussed below.

[0048] It is contemplated that the skilled artisan may use anyconventional procedure for removing a particular surfactant or moreparticularly, Zwittergent® from the mixture. For example, it iscontemplated that the surfactant may removed by, for example,conventional ion exchange chromatography or, more preferably by organicsolvent extraction. Furthermore, it is contemplated that the surfactantmay be removed prior to, subsequent to, or during the removal of theprecipitate from the mixture.

[0049] In a preferred embodiment, Zwittergent® surfactant is removedfrom the mixture simultaneously with the precipitate by organic solventextraction. For example, following the production of the precipitate,the resulting mixture may be extracted with an organic solvent thatdissolves surfactant and is compatible with amebocyte lysate. In anexemplary protocol, organic solvent is added to the mixture of amebocytelysate and surfactant, and the combination thoroughly mixed. Afterextraction, the combination may separate to produce a three phase systemcomprising an organic phase, an interface of precipitate, and an aqueousphase. When an organic solvent, for example, chloroform is employed, theresulting aqueous phase is located above the organic phase, with theprecipitate residing at the interface of the organic and aqueous phases.

[0050] It is contemplated that any organic solvent which dissolves thesurfactant and which is compatible with amebocyte lysate (i.e., does notimpair the Factor C cascade) may be used to remove the surfactant. Also,it has been noted that certain organic solvents may be used to remove orinactivate lysate inhibitors, which when removed or inactivated enhancethe sensitivity of the resulting lysate to endotoxin. See, for example,U.S. Pat. Nos. 4,107,077 and 4,279,774, the disclosures of which areincorporated herein by reference. Accordingly, it may be preferable, butnot essential that the organic solvent used to remove the surfactantalso remove or inactive the lysate inhibitors. Preferred solventsinclude, without limitation, chloroform, iodoform, bromoform, loweralkyl halides such as methyl bromide, methyl chloride, methyl iodide,ethyl chloride, ethyl iodide, propyl chloride, propyl bromide and propyliodide, ethylene dichloride, methylene dichloride, benzene,monohalobezenes such as chlorobenzene, bromobenzene and iodobenzene,lower alkyl ethers such as dimethyl ether and diethyl ether, carbontetrachloride, trichioroethane, trichloroethylene, toluene and hexane.Choice of the optimal organic solvent for a particular surfactant may bedetermined by routine experimentation

[0051] In a preferred embodiment, when using the sulfobetaine detergentZwittergent® 3-14, chloroform is the preferred organic solvent for usein the organic extraction. Chloroform is not only compatible with thelysate and capable of dissolving Zwittergent® 3-14, but also is capableof removing and/or inactivating known LAL inhibitors in amebocytelysate. It is understood that the amount of detergent removed can bealtered, thereby altering the endotoxin sensitivity of the lysate, byvarying the chloroform to lysate ratio during organic solventextraction.

[0052] Following extraction, the resulting organic and aqueous phasesare allowed to separate. Separation may be speeded up by centrifugation.For example, phase separation may be speeded up by centrifugation atabout 500G to about 2,000G, most preferably about 1,200G. Followingcentrifugation, the aqueous phase is harvested. For example, whenchloroform is used, the upper aqueous phase may be harvested bydecanting the aqueous phase thereby leaving behind the interfacialmaterial and the lower chloroform organic phase. The decanted upperaqueous phase, preferably is subjected to a second round ofcentrifugation, for example, at about 5,000G to about 9,000G, mostpreferably about 7,000G. The resulting aqueous phase then is separatedand harvested from residual interfacial material and/or organic phase,and either stored or formulated as desired. It is appreciated, however,that the optimal number and extent of the centrifugation steps for aparticular system may be determined by routine experimentation.

[0053] The resulting upper aqueous phase can then stored at reducedtemperature or formulated immediately. For example, when frozen lysatemay retain activity indefinitely, whereas, when stored at 2-8° C., thelysate may retain activity for several weeks. The reduction in Factor Gactivity of the resulting amebocyte lysate preparations can bedetermined using any of the techniques described hereinbelow.

Enhancement of the Sensitivity of Amebocyte Lysate Preparations toEndotoxin

[0054] As discussed hereinabove, it has been discovered that thesensitivity to endotoxin of an amebocyte lysate preparation havingreduced Factor G activity may be enhanced by the addition of anexogenous (1→3)-β-D glucan. An increase in sensitivity is understood tomean that the lysate with additive reacts faster to produce a product atlower endotoxin concentrations than lysate without additive. Inparticular, (1→3)-β-D glucan can be added to the lysate preparation inan amount sufficient to enhance the endotoxin sensitivity of the lysatepreparation relative to a similar amebocyte lysate preparation withoutthe exogenously added (1→3)-β-D glucan. It is understood, however, thatthe same amount of (1→3)-β-D glucan when added to crude amebocyte lysatelikely would induce the production of a coagulin gel via the Factor Gcascade. In effect, during the practice of this particular embodiment ofthe invention, surprisingly a substrate or initiator of the now reducedor depleted Factor G cascade is added to the amebocyte lysatepreparation of the invention. It is contemplated that this type ofenhancement may occur with any lysate depleted of Factor G activity(e.g., wherein Factor G is removed from lysate or wherein the lysatecontains Factor G inhibitors).

[0055] The identification of suitable (1→3)-β-D glucans as well as theidentification of optimal concentrations of (1→3)-β-D glucans forenhancing endotoxin sensitivity may be determined by routineexperimentation using the methodologies described hereinbelow. Withregard to the type of useful (1→3)-β-D glucans, it is contemplated thatany (1→3)-β-D glucan that induces a Factor G mediated cascade in crudeamebocyte lysate can be used in the practice of this aspect of theinvention. Currently preferred (1→3)-β-D glucans include, withoutlimitation, natural polysaccharides obtained from cell walls of, forexample, various bacteria (for example, Alcaligenes genus, andAgrobacterium genus), yeasts (for example, shiitake) with specificexamples of natural polysaccharides including, for example, curdlan,pachyman, scleratan, leutinan, schizophylan, and coriolan. Other naturalpolysaccharides include storage polysaccharides of algae, for example,brown algae, Euglena, diatoma, with specific examples of storagepolysaccharides including, for example, laminaran, laminarin, andparamilon. Preferred (1→3)-β-D glucans also include, for example,polysaccharide derivatives in which at least one group selected from acarboxymethyl group, a carboxyethyl group, a methyl group, ahydroxyethyl group, a hydroxypropyl group, and a sulfopropyl group, isintroduced into a natural polysaccharide or storage polysaccharide usingconventional methodologies well known in the art. See, for example,Munio Kotake “Daiyukikagaku” Vol. 19 7^(th) ed. Asakura Shoten, May 10(1967) pp. 70-101; A. E. Clarke et al. (1967) Phyto-chemistry 1:175-188; and T. Sasaki et al. (1967) Europ. J. Cancer, 15: 211-215.Other naturally occurring polysaccharides may be derived from cottonwool, for example, in cotton wool extracts, and certain cellulose-basedfilters used in the processing of medicinals. See, for example,Roslansky et al. (1991) J. Clin. Micro. 29: 2477; Henne et al. (1984)Artif. Organs 8: 299; and Ikemura et al. (1989) J. Clin. Micro. 27:1965. Furthermore, it is contemplated that the aforementionedpolysaccharides and derivatives thereof may be used either alone or incombination with others to enhance endotoxin sensitivity.

[0056] Although it is contemplated that any amount of (1→3)-β-D glucanthat enhances the sensitivity of the amebocyte lysate to the endotoxinrelative to similar amebocyte lysate without the exogenously added(1→3)-β-D glucan can be used in the practice of this aspect of theinvention, the optimal amount of exogenous (1→3)-β-D glucan forenhancing the endotoxin-specific cascade in a particular lysate can bedetermined by routine experimentation. For example, the optimalconcentration can be determined by adding different amounts of aparticular (1→3)-β-D glucan to crude amebocyte lysate, wherein theoptimal amount is the amount of (1→3)-β-D glucan that induces thefastest coagulin clot or color formation in crude amebocyte lysate. See,for example, Examples 4, 5 and 6, disclosed hereinbelow. These assayprotocols are considered to be exemplary, and it is understood that theskilled artisan may use a variety of assays, for example, gel-clot,kinetic turbidimetric, end-point turbidimetric or chromogenic assays, todetermine the optimal amount of (1→3)-β-D glucan to be added to thelysate of interest.

[0057] Once the optimal concentration of a particular (1→3)-β-D glucanfor enhancing endotoxin sensitivity has been determined, then the sameof amount of the (1→3)-β-D glucan can then be added to an amebocytelysate preparation having reduced or eliminated Factor G activitythereby to enhance the endotoxin sensitivity of the resulting mixture.It is contemplated that the endotoxin sensitivity of any amebocytelysate having reduced Factor G activity may be enhanced by the additionof (1→3)-β-D glucan. For example, it is contemplated that the endotoxinsensitivity of any amebocyte lysate having reduced Factor G activity maybe enhanced by the addition of exogenous (1→3)-β-D glucan.

Formulation of Amebocyte Lysate

[0058] The resulting lysate may be formulated using conventionalmethodologies well known and thoroughly discussed in the art. See, forexample, R. B. Prior, Ed., (1990), “Clinical Applications of the LimulusAmebocyte Lysate Test”, CRC Press, and U.S. Pat. No. 4,322,217, thedisclosures of which are incorporated herein by reference. Methods forenhancing sensitivity of amebocyte lysate may include, withoutlimitation, the aging of crude amebocyte lysate, adjustment of pH,adjustment of divalent cation concentration, adjustment of coagulogenconcentration, chloroform extraction, and the addition of serum albumin,biocompatible buffers and/or biological detergents.

[0059] For example, typical formulation additives may include, withoutlimitation, about 100-300 mM NaCl, about 10-100 mM divalent cations(e.g., Mg²⁺ or Ca²⁺), biocompatible buffers, e.g., Tris, to give a finalpH of about 6.0 to 8.0, and, if the lysate is to be freeze dried, thensugars, e.g., mannitol or dextran. It is contemplated that, the choiceof appropriate formulation additives may be determined by routineexperimentation.

[0060] Lysate, once formulated, typically is lyophilized for long termstorage. The lyophilized amebocyte lysate formulations may bereconstituted prior to use by the addition of, for example, pyrogen-freewater, or any other pyrogen-free biocompatible buffer.

Methods for Measuring Bacterial Endotoxins

[0061] It is contemplated that the amebocyte lysate preparations of theinvention may be used to detect and/or quantitate the amount ofendotoxin in any sample of interest. For example, it is contemplatedthat the lysate may be used to detect and/or measure the concentrationof endotoxin in any pharmaceutical preparation, for example, anorganically produced drug or a recombinantly produced protein, and/orany medical device of interest. In addition, the lysate may be used todetect and/or measure the concentration of endotoxin in biologicalsamples of, for example, blood, serum, plasma, urine, semen, asciticfluid, peritoneal fluid, sputum, breast exude, and spinal fluid.

[0062] It is contemplated that the amebocyte lysate of the invention maybe used to detect and/or quantitate a bacterial endotoxin in a sampleusing any lysate-based assay now known or later developed that detectsone or more products of the Factor C cascade. It is contemplated,however, that the amebocyte lysate preparation of the invention may beused to advantage in any conventional gel-clot, end-point turbidimetric,kinetic turbidimetric, or chromogenic assay, known and/or used in theart. The particulars of each of these four types of assays are describedbelow.

(i) Gel-Clot Assay

[0063] This technique is described in Prior, R. B., Ed., supra, pp.28-34, the disclosure of which is incorporated by reference herein, and,therefore, is not described in detail herein. Briefly, the gel-clotassay comprises the steps of (i) mixing amebocyte lysate preparationwith the sample to be analyzed, (ii) incubating the resulting mixture ata temperature of 0° to 40° C., preferably 25° to 40° C., for apredetermined time, for example, one hour, and (iii) visually inspectingwhether or not a gel-clot has been produced.

(ii) End Point Turbidimetric Assay

[0064] This technique is described in Prior, R. B., Ed., supra, pp.28-34 and, therefore, is not described in detail herein. Briefly, theend point turbidimetric assay comprises the steps of (i) mixingamebocyte lysate preparation with a sample to be investigated, (ii)incubating the resulting mixture at a temperature of 0° to 40° C.,preferably 25° to 40° C., for a predetermined time, and (iii) measuringthe increase in turbidity as a result of coagulation, if any, using aconventional coagulometer, nepherometer, spectrophotometer, or the like.

(iii) Kinetic Turbidimetric Assay

[0065] This technique is described in Prior, R. B., Ed., supra, pp.28-34 and, therefore, is not described in detail herein. Briefly, thekinetic turbidimetric assay comprises the steps of (i) mixing amebocytelysate preparation with a sample to be investigated, (ii) incubating theresulting mixture at a temperature of 0° to 40° C., preferably 25° to40° C., over a predetermined time range, and (iii) measuring a timerequired for either a turbidity change caused by coagulation to reach apreselected value or a ratio in change of the turbidity, using aconventional coagulometer, nepherometer, spectrophotometer, or the like.

(iv) Chromogenic Assay

[0066] This technique is described in Prior, R. B., Ed., supra, pp.28-34, and U.S. Pat. Nos.: 4,301,245; 4,717,658; and 5,310,657, thedisclosures of which are incorporated herein by reference and,therefore, is not described in detail herein. Briefly, the chromogenicassay comprises the steps of (i) mixing amebocyte lysate preparationwith a sample to be investigated, (ii) incubating the resulting mixtureat a temperature of 0° to 40° C., preferably 25° to 40° C., for apredetermined time, then, if necessary, adding a reaction inhibitor, and(iii) measuring a substance released by protease activity from thesynthetic substrate calorimetrically, or the like.

EXAMPLES

[0067] Practice of the invention will be more fully understood from thefollowing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

Example 1 Preparation and Characterization of Amebocyte Lysate Depletedof Factor G Activity

[0068] This example describes a preferred method for producing anamebocyte lysate having reduced Factor G activity. Throughout thefollowing procedure, all reagents and apparatus, where appropriate, areproduced or treated to be pyrogen free. Such methodologies are wellknown in the art and, therefore, are not discussed in detail herein. Forexample, apparatus may be made pyrogen-free by baking in an oven at≧200° C. for four hours, and reagents may be made pyrogen-free bytreatment by ultra filtration (≦20 kD cut off), oxidation with peroxide,or treatment with NaOH.

[0069] Crude amebocyte lysate was prepared by harvesting hemolymph fromhorseshoe crab. The resulting hemolymph was centrifuged to produce anamebocyte pellet. The amebocytes then were reharvested, rerinsed andrecentrifuged. After second rinsing and harvesting steps, the resultingamebocytes were lysed by osmotic shock, and the resulting crudeamebocyte lysate stored at 2-8° C. until further use.

[0070] Thereafter, Zwittergent® 3-14 was added stepwise to the crudelysate to a final concentration of 0.12% (w/v). The resulting solutionwas stored at 2-8° C. for 8-24 hours. Thereafter, the solution was mixedwith chloroform (4-5 parts lysate to 1 part chloroform) by gentlestirring for 10-30 minutes at 2-8° C. The resulting mixture was thencentrifuged at 1,200 G for 15 minutes, whereupon the resulting upperaqueous and lower organic phases were separated by interfacialprecipitate material. The aqueous phase then was harvested by decanting,and recentrifuged at 7,000 G for at least 30 minutes to achieve clarity.After centrifugation, the resulting aqueous phase was decanted from theresidual organic phase and interfacial material. The extent of detergentextraction was estimated by thin layer chromatography (TLC). Briefly,samples of chloroform extract and lysate were applied to a TLC plate(Whatman PE SIL GLUV), developed with methanol:water (10:1(v/v)), andthe detergent visualized with UV light. By this procedure, no residualdetergent was detectable in the lysate.

[0071] Fractionation of the crude amebocyte lysate, the precipitateproduced by the surfactant, and the supernatant containing the amebocytelysate by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) suggests that Factor G is actually contained in theprecipitate and, therefore, is removed from the lysate. Densitometricprofiles of each lane of the resulting Coomassie Blue stained SDS-PAGEgel are shown in FIG. 3.

[0072] In FIG. 3, lane 1 represents the densitometric profile ofmolecular weight markers, wherein peak 2 represents a protein having amolecular weight of 66 kD, peak 3 represents a protein having amolecular weight of 45 kD, peak 4 represents a protein having amolecular weight of 36 kD, peak 5 represents a protein having amolecular weight of 29 kD, peak 6 represents a protein having amolecular weight of 24 kD, peak 7 represents a protein having amolecular weight of 20 kD, peak 8 represents a protein having amolecular weight of 14.2 kD, and peak 9 represents a protein having amolecular weight of 6.5 kD.

[0073] In FIG. 3, lane 2 represents a fractionated sample of crudeamebocyte lysate wherein peaks 12 and 15 apparently represent the twosubunits of Factor G (one having a molecular weight of about 76 kD andthe other having a molecular weight of about 36 kD). Lane 3 represents afractionated sample of amebocyte lysate supernatant following detergentprecipitation. According to lane 3, it appears that the supernatant isdepleted of the 76 kD and 36 kD Factor G subunits. Lane 4 represents afractionated sample of the lysate precipitate. According to lane 4, itappears that the precipitate contains the 76 kD and 36 kD subunits ofFactor G (peaks 29 and 33/34, respectively). According to theseprofiles, it appears that Factor G was actually precipitated from thelysate by the surfactant.

Example 2 Formulation of Amebocyte Lysate Depleted of Factor G Activity

[0074] Following preparation of the amebocyte lysate preparation of theinvention, the resulting lysate was formulated as follows: PERCENTAGECOMPONENTS OF COMPONENTS Extracted Limulus Amebocyte Lystate 37% Water 8% 0.4 M Mg²⁺/0.6 M Tris-HEPES Buffer 10% 6% Dextran  6% 3% NaCl 39%Laminarin 0.000004%-0.000013%

[0075] The resulting formulation was stored at 2-8° C. until use.

Example 3 Reactivity of Crude Amebocyte Lysate and Lysate Depleted ofFactor G Activity with Bacterial Endotoxin

[0076] This example demonstrates that amebocyte lysate of the inventionstill reacts with bacterial endotoxin, as determined by the gel-clotassay. Three batches of amebocyte lysate having reduced Factor Gactivity (referred to as L4941LB, L1081LB, L1711LB) as produced by themethod of Example 1 and formulated as described in Example 2 were testedfor endotoxin reactivity using a gel-clot assay. Batches L4941LB andL1081LB were also formulated with 0.4% bulking protein and 0.01%Zwittergent® 3-14.

[0077] The endotoxin reactivity of each batch of lysate was determinedsimultaneously with a control batch of lysate, referred to as referencelysate lot 13, obtained from the USFDA. The endotoxin standard used ineach determination was obtained from USFDA, and is referred to herein asEC-6. Briefly, a standard solution of EC-6 endotoxin was prepared.Thereafter, endotoxin was added to the reference lysates to give a finalendotoxin concentration of 0.5, 0.25, 0.125, 0.06, or 0.03 EU/mL.Similarly, endotoxin was added to the test lysates to give a final toxinconcentration of 0.06, 0.03, 0.015, or 0.007 EU/mL. After mixing, theresulting mixtures were incubated at 37° C. for one hour, after whichthe presence or absence of a clot was noted. All the samples weretreated the same.

[0078] In order to assure the integrity of the assay, the assays wereperformed in a specific order. For example, a first batch of fourreference lysate samples (denoted by test number 1 in Tables 1, 3, and5) was analyzed first, then a batch of ten lysate test samplesformulated as described in Example 2 (denoted by vial numbers 1-10 inTables 2,4, and 6) was analyzed, and finally a second batch of fourreference lysate samples (denoted by test number 2 in Tables 1, 3, and5) was analyzed.

[0079] Each test sample was analyzed side-by-side with a referencelysate. Accordingly, data relating to the reactivity of reference lysate13 (Table 1) was derived contemporaneously with data relating to thereactivity of the test lysate batch number L1711LB (Table 2). Accordingto Table 1, the reactivity of reference lysate 13 had an endpoint value(E.Pt.) of 0.06 EU/mL for the assay (i.e., within a recognizedacceptable level). According to Table 2, the reactivity of test lysateL1711LB (10 samples) had an endpoint value estimated at 0.06 EU/mL.Accordingly, the test lysate L1711LB was level of sensitivity. TABLE 1Reference Lysate Lot 13, Endotoxin Lot EC-6 TEST 0.5 0.25 0.125 0.060.03 E.Pt. 1 + + + + − 0.06 1 + + + + − 0.06 1 + + + + − 0.06 1 + + + +− 0.06 2 + + + + − 0.06 2 + + + + − 0.06 2 + + + + − 0.06 2 + + + + −0.06

[0080] TABLE 2 Test Lysate Lot L 1711LB, Endotoxin Lot EC-6 VIAL 0.1250.06 0.03 0.015 E.Pt. 1 + + − − 0.06 2 + + − − 0.06 3 + + − − 0.06 4 + +− − 0.06 5 + + − − 0.06 6 + + − − 0.06 7 + + − − 0.06 8 + + − − 0.069 + + − − 0.06 10 + + − − 0.06

[0081] Similarly, data relating to the reactivity of reference lysate 13(Table 3) was derived contemporaneously with data relating to thereactivity of the test lysate batch number L1081LB (Table 4). Accordingto Table 3, the reactivity of reference lysate 13 had an endpoint value(E.Pt.) of 0.06 EU/mL for the assay (i.e., within a recognizedacceptable level). According to Table 4, the reactivity of test lysateL1081LB (10 samples) had an endpoint value estimated at 0.015 EU/mL.Accordingly, the test lysate L1081LB was more sensitive to endotoxinthan the reference lysate. TABLE 3 Reference Lysate Lot 13, EndotoxinLot EC-6 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt. 1 + + + + − 0.06 1 + + + +− 0.06 1 + + + + − 0.06 1 + + + + − 0.06 2 + + + + − 0.06 2 + + + + −0.06 2 + + + + − 0.06 2 + + + + − 0.06

[0082] TABLE 4 Test Lysate Lot L1081LB, Endotoxin Lot EC-6 VIAL 0.060.03 0.015 0.007 E.PT. 1 + + + − 0.015 2 + + + − 0.015 3 + + + − 0.0154 + + + − 0.015 5 + + + − 0.015 6 + + + − 0.015 7 + + + − 0.015 8 + + +− 0.015 9 + + + − 0.015 10 + + + − 0.015

[0083] In addition, data relating to the reactivity of reference lysate13 (Table 5) was derived contemporaneously with data relating to thereactivity of the test lysate batch number L4941LB (Table 6). Accordingto Table 5, the reactivity of reference lysate 13 had an endpoint value(E.Pt.) of 0.06 EU/mL for the assay (i.e., within a recognizedacceptable level). According to Table 6, the reactivity of test lysateL4941LB (10 samples) had an endpoint value estimated at 0.03 EU/mL.Accordingly, the test lysate L4941LB was more sensitive to endotoxinthan the reference lysate. TABLE 5 Reference Lysate Lot 13, EndotoxinLot EC-6 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt. 1 + + + + − 0.06 1 + + + +− 0.06 1 + + + + − 0.06 1 + + + + − 0.06 2 + + + + − 0.06 2 + + + + −0.06 2 + + + + − 0.06 2 + + + + − 0.06

[0084] TABLE 6 Test Lysate Lot L4941LB, Endotoxin Lot EC-6 VIAL 0.060.03 0.015 0.007 E.PT. 1 + + + − 0.015 2 + + + − 0.015 3 + + − − 0.034 + + − − 0.03 5 + + + − 0.015 6 + + − − 0.03 7 + + − − 0.03 8 + + − −0.03 9 + + + − 0.015 10 + + − − 0.03

[0085] All three batches of lysate, L4941LB, L1081LB and L1711LB met the24 hour negative control requirements as required by the USFDA.

Example 4 Reactivity of Crude Amebocyte Lysate and Lysate Depleted ofFactor G Activity with Laminarin

[0086] This example demonstrates that Limulus amebocyte lysates producedby the method of the invention, unlike crude lysates, are insensitive tothe presence of laminarin in the sample. In addition, this exampledefines the optimal amount of laminarin (as determined from the crudelysate sample) that may be exogenously added to the lysate preparationof the invention, thereby to enhance the sensitivity of the lysatepreparation to endotoxin.

[0087] A standard solution of laminarin was produced by dissolving 1.0 gof laminarin in 100 ml of 5 mM NaOH to produce a 10 mg/mL solution. Theresulting solution was autoclaved at 121° C. for one hour. Afterautoclaving, the pH was adjusted to 7.0 with 0.1M Tris buffer. Thissolution was then diluted in pyrogen-free water to give a finallaminarin stock solution of 0.2 mg/mL. A dilution series of laminarinwas produced, and the reactivity of crude lysate and the lysatepreparation of the invention to laminarin was determined both bygel-clot and kinetic-turbidimetric assays.

I. Gel-Clot Assay

[0088] Gel-clot assays were performed by combining either crude lysateor lysate preparations of the invention with different amounts oflaminarin. Crude lysate (batch K2222L) was formulated essentially asdescribed in Example 2, except that the “Extracted Limulus AmebocyteLysate” was replaced with crude lysate and the concentration oflaminarin was varied between samples. The lysate preparations of theinvention (batch L4941LB, batch L1081LB, and batch L1711LB) wereformulated essentially as described in Example 3. except theconcentration of laminarin was varied between samples. The samples wereincubated at 37° C., and the presence or absence of clots determinedafter one hour. Each experiment was performed in duplicate, and theresults summarized in Table 7. TABLE 7 LAMINARIN Conc. K2222L L4941LBL1081LB L1711LB Dilution mg/mL 0.03 0.03 0.015 0.06  1    50 4.00E−03−    − −    − −    − −    −  2   100 2.00E−03 −    − −    − −    −−    −  3   200 100E−03 −    − −    − −    − −    −  4  5  6  7  8   400  800   1600   3200   6400 5.00E−04 2.50E−04 1.25E−04 6.25E−05 3.13E−05

−    −−    −−    −−    −−     #− −    −−    −−    −−    −−    −−    −−    −−    −−    −−    − 9 12800 1.56E−05 −    − −    − −    −−    − 10 25600 7.81E−06 −    − −    − −    − −    − 11 51200 3.91E−06−    − −    − −    − −    − 12 102400 1.95E−06 −    − −    − −    −−    − 13 204800 9.77E−07 −    − −    − −    − −    − 14 409600 4.88E−07−    − −    − −    − −    − 15 819200 2.44E−07 −    − −    − −    −−    − 16 1638400 1.22E−07 −    − −    − −    − −    − 17 32768006.10E−08 −    − −    − −    − −    − 18 6553600 3.05E−08 −    − −    −−    − −    −

[0089] The results in Table 7 indicate that none of the batches producedby the method of invention (i.e., L4941LB, L1081LB and L1711LB) werereactive with exogenously added laminarin. In contrast, the batch ofcrude lysate (K2222L) was reactive with liminarin over the dilutionrange of 400 through 6400 in a gel-clot assay.

II. Kinetic-Turbidimetric Assay

[0090] Reactions were initiated by combining either formulated crudelysate (batch K2222L) or formulated lysate preparations of the invention(batch L4941LB, batch L1081LB, and batch L1711LB) with different amountsof laminarin. The assay was performed using a Biotek elx-808 incubatingmicroplate reader in accordance with the manufacturers instructions.Each assay was run, at least, in duplicate. During the assay, thesamples were incubated at 37±0.2° C., and data was collected over aperiod of one hour.

[0091] The assay was performed simultaneously using additional lysatesamples incubated in the presence of known amounts of endotoxinstandards. Endotoxin standards were used to generate a standard curvecovering a 4-log range, for example, from 50 to 0.005 EU/mL. After datacollection, the data was analyzed using Biotek Kc3-cre kinetic software(available from Charles River Endosafe, Charleston, S.C.). The sampleconcentrations were interpreted as “endotoxin values” by the instrumentand were represented by the units of EU/mL because they were estimatedfrom the endotoxin standard curve. The resulting concentration valuesreflect the reactivity of the lysate to laminarin, and are referred toas “endotoxin equivalent values”.

[0092] By plotting the laminarin endotoxin equivalent values versusdilution factor, it is possible to generate a bell-shaped curve withcrude lysate. The peak of the curve provides the optimal amount oflaminarin which can be added to amebocyte lysate depleted of Factor Gactivity, thereby to enhance the sensitivity of the lysate.

[0093] The results of the experiment are shown in FIG. 4. According toFIG. 4, none of the batches produced by the method of the invention(i.e., L4941LB, L1081LB and L1711LB) were reactive with exogenouslyadded laminarin. In contrast, the batch of crude lysate (K2222L) wasreactive with laminarin over the dilution range of 100 through 51200,with the dilution value of 3200 producing the maximal endotoxinequivalent value. Accordingly, this value represents the optimal amountof laminarin to be added to amebocyte lysate depleted of Factor Gactivity to provide maximal sensitivity to endotoxin in a kineticturbidimetric assay.

Example 5 Reactivity of Crude Amebocyte Lysate and Lysate Depleted ofFactor G Activity with LAL Reactive Material

[0094] This example demonstrates that Limulus amebocyte lysates producedby the method of the invention, unlike crude lysates, are insensitive tothe presence in the sample of LAL reactive material (LAL-RM) produced byrinsing a cellulose-acetate filter with water. In addition, this exampledefines the optimal amount of this LAL-RM (as determined from the crudelysate sample) that may be exogenously added to the lysate preparationof the invention, thereby to enhance the sensitivity of the lysatepreparation to endotoxin.

[0095] LAL-RM was prepared by passing one liter of pyrogen-free waterthrough a conventional sterile, non-pyrogenic cellulose acetate hollowfiber membrane adapted for use in renal dialysis. The resulting rinsethen was used to create a dilution series of cellulose acetate rinse.The reactivity of crude lysate and the lysate preparation of theinvention to the cellulose acetate rinse was determined both by gel-clotand kinetic-turbidimetric assays.

I. Gel-Clot Assay

[0096] The gel-clot assays were performed as described in Example 4Iabove, except in the formulations laminarin was replaced by celluloseacetate rinse. The assays were performed by combining either formulatedcrude lysate (batch K2222L) or formulated lysate preparations of theinvention (batch L4941LB, batch L1081LB, and batch L1711LB) withdifferent amounts of cellulose acetate rinse. The samples were incubatedat 37° C., and the presence or absence of clots assessed after one hour.Each experiment was performed in duplicate and the results summarized inTable 8. TABLE 8 Cellulose Acetate Rinse Conc. K2222L L4941LB L1081LBL1711LB Dilution mg/mL 0.03 0.03 0.015 0.06  1  2  3  4  5  6  7  8   50   100   200   400   800   1600   3200   6400 4.00E−03 2.00E−031.00E−03 5.00E−04 2.50E−04 1.25E−04 6.25E−05 3.13E− #05

−    −−    −−    −−    −−    −−    −−    −−    −−    −−    −−    −−    −−    −−    −−     #−−    −−    −−    −−    −−    −−    −−    −−    −−    −  9  12800 1.56E−05−    − −    − −    − −    − 10  25600 7.81E−06 −    − −    − −    −−    − 11  51200 3.91E−06 −    − −    − −    − −    − 12  1024001.95E−06 −    − −    − −    − −    − 13  204800 9.77E−07 −    − −    −−    − −    − 14  409600 4.88E−07 −    − −    − −    − −    − 15  8192002.44E−07 −    − −    − −    − −    − 16 1638400 1.22E−07 −    − −    −−    − −    − 17 3276800 6.10E−08 −    − −    − −    − −    − 18 65536003.05E−08 −    − −    − −    − −    −

[0097] The results of Table 8 indicate that none of the batches producedby the method of the invention (i.e., L4941LB, L1081LB and L1711LB) werereactive with exogenously added cellulose acetate rinse as determined bygel-clot assay. In contrast, the batch of crude lysate (K2222L) wasreactive with cellulose acetate over the dilution range of 50 through6400 in a gel-clot assay.

II. Kinetic-Turbidimetric Assay

[0098] This assay was performed as described in Example 4II above,except in the formulations laminarin was replaced by cellulose acetaterinse. Reactions were initiated by combining either crude lysate (batchK2222L) or lysate preparations of the invention (batch L4941LB, batchL1081LB, and batch L1711LB) with different amounts of cellulose acetaterinse. The results of the assay are presented in FIG. 5. According toFIG. 5, none of the batches produced by the method of the invention(i.e., L4941LB, L1081LB and L1711LB) were reactive with exogenouslyadded cellulose acetate. In contrast, the batch of crude lysate (K2222L)was reactive with cellulose acetate over the dilution range of 50through 102400 with the dilution value of 400 producing the maximalendotoxin equivalent value. Accordingly, this value represents theoptimal amount of cellulose acetate to be added to amebocyte lysatedepleted of Factor G activity to provide maximal sensitivity toendotoxin in a kinetic turbidimetric assay.

Example 6 Reactivity of Crude Amebocyte Lysate and Lysate Depleted ofFactor G Activity with Cotton Extract

[0099] This example demonstrates that Limulus amebocyte lysates producedby the method of the invention, unlike crude lysates, are insensitive tothe presence in the sample of cotton extract. In addition, this exampledefines the optimal amount of cotton extract (as determined from thecrude lysate sample) that may be exogenously added to the lysatepreparation of the invention, thereby to enhance the sensitivity of thelysate preparation to endotoxin.

[0100] Cotton extract was prepared as follows. Approximately, 1 gram ofcotton (about 4 one inch cotton balls) was added to 40 ml of 1 N NaOH ina 50 ml polystyrene conical tube. Thereafter, the mixture was incubatedat room temperature (about 20° C.) for 10 days. Prior to use, thesolution was decanted from the cotton into a fresh, pyrogen-free tube(baked 200° C., 6 h) and neutralized with HCl to produce a stocksolution of cotton extract. Further dilutions were made in pyrogen-freewater (available from Charles River Endosafe, Charleston, S.C.). Thereactivity of crude lysate and the lysate preparation of the inventionto cotton extract were determined both by gel-clot andkinetic-turbidimetric assays.

I. Gel-Clot Assay

[0101] The gel-clot assays were performed as described in Example 4Iabove, except in the formulations laminarin was replaced by cottonextract. The assays were performed by combining either formulated crudelysate (batch K2222L) or formulated lysate preparations of the invention(batch L4941LB, batch L1081LB, and batch L1711LB) with different amountsof cotton extract. The samples were incubated at 37° C., and thepresence or absence of clots determined after one hour. Each experimentwas performed in duplicate and the results summarized in Table 9. TABLE9 LAMINARIN Conc. K2222L L4941LB L1081LB L1711LB Dilution mg/mL 0.030.03 0.015 0.06  1    50 4.00E−03 /    / /    / /    / /    /  2  3  4 5   100   200   400   800 2.00E−03 1.00E−03 5.00E−04 2.50E−04

−    −−    −−    −−    − −    −−    −−    −−    # −−    −−    −−    −−    −  6   1600 1.25E−04 −    − −    − −    − −    − 7   3200 6.25E−05 −    − −    − −    − −    −  8   6400 3.13E−05 −    −−    − −    − −    −  9  12800 1.56E−05 −    − −    − −    − −    − 10 25600 7.81E−06 −    − −    − −    − −    − 11  51200 3.91E−06 −    −−    − −    − −    − 12  102400 1.95E−06 −    − −    − −    − −    − 13 204800 9.77E−07 −    − −    − −    − −    − 14  409600 4.88E−07 −    −−    − −    − −    − 15  819200 2.44E−07 −    − −    − −    − −    − 161638400 1.22E−07 −    − −    − −    − −    − 17 3276800 6.10E−08 −    −−    − −    − −    − 18 6553600 3.05E−08 −    − −    − −    − −    −

[0102] The results in Table 8 indicate that none of the batches producedby the method of the invention (i.e., L4941LB, L1081LB and L1711LB) werereactive with exogenously added cotton extract. In contrast, the batchof crude lysate (K2222L) was reactive with cotton extract over thedilution range of 100 through 800 in a gel-clot assay.

II. Kinetic-Turbidimetric Assay

[0103] This assay was performed as described in Example 4II above,except in the formulations laminarin was replaced by cotton extract.Reactions were initiated by combining either crude lysate (batch K2222L)or lysate preparations of the invention (batch L4941LB, batch L1081LB,and batch L1711LB) with different amounts of cotton extract. The resultsof the assay are presented in FIG. 6. According to FIG. 6, none of thebatches produced by the method of the invention (i.e., L4941LB, L1081LBand L1711LB) were reactive with exogenously added cotton extract. Incontrast, the batch of crude lysate (K2222L) was reactive with cottonextract over the dilution range of from at least 100 through 3200.According to the results, it appears that cotton extract also may beused to enhance the sensitivity of Factor G depleted lysate toendotoxin.

Example 7 Endotoxin Sensitivity of Amebocyte Lysate Depleted of Factor GActivity With and Without (1→3)-β-D Glucan

[0104] This example demonstrates that the sensitivity to endotoxin of anamebocyte lysate depleted of Factor G activity can be enhanced by theaddition of exogenous (1→3)-β-D glucan.

[0105] Briefly a batch of amebocyte lysate prepared in accordance withExample 1 was formulated essentially as described in Example 2. Acontrol batch was formulated without the exogenously added laminarin.The reactivities of each lysate to different amounts of endotoxin weremeasured by kinetic-turbidimetric assay, essentially as described inExample 4II. Briefly, different amounts of endotoxin (USFDA lot EC-6)were added to each sample of lysate and the reaction end pointdetermined by Bio-tek KC3-cre kinetic software. The results are shown inFIG. 7. The circles represent samples of lysate not supplemented withlaminarin (control samples), and the boxes represent samples of lysatesupplemented with laminarin (test samples).

[0106] According to FIG. 7, at high concentrations of endotoxin (i.e.,about 50 EU/ml) there appears to be little difference in reaction timebetween lysate supplemented with laminarin and lysate not supplementedwith laminarin. However, at low concentrations of endotoxin (i.e., about≦0.05 EU/mL), the lysate supplemented with laminarin reactedsignificantly faster with endotoxin than lysate not supplemented withlaminarin. Accordingly, these results demonstrate that it is possible toenhance the sensitivity of an amebocyte lysate depleted of Factor Gactivity by the addition of exogenous (1→3)-β-D glucan.

Equivalents

[0107] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Theforegoing embodiments are therefore to be considered in all respectsillustrative rather than limiting on the invention described herein.Scope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are intended to beembraced therein.

What is claimed is:
 1. A method of producing an improved amebocytelysate preparation having reduced Factor G activity, the methodcomprising the steps of: (a) admixing crude amebocyte lysate with asurfactant in an amount sufficient to produce a solution containing aprecipitate; and (b) separating said precipitate from said solutionthereby to produce an amebocyte lysate preparation which is lessreactive with (1→3)-β-D glucan than said crude amebocyte lysate.
 2. Themethod of claim 1, wherein said amebocyte lysate preparation is reactivewith a bacterial endotoxin.
 3. The method of claim 1 or 2, wherein saidprecipitate contains Factor G.
 4. The method of claim 1 or 2, whereinsaid surfactant is a zwitterionic surfactant.
 5. The method of claim 4,wherein said zwitterionic surfactant is a sulfobetaine-type surfactant.6. The method of claim 5, wherein said sulfobetaine-type surfactant isselected from the group consisting of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
 7. The method of claim 5,wherein said sulfobetaine-type surfactant is n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
 8. The method of claim 1,comprising the additional step of removing said surfactant from saidsolution.
 9. The method of claim 8, wherein said surfactant is removedby organic solvent extraction.
 10. The method of claim 9, wherein saidorganic solvent is chloroform.
 11. A method of producing an improvedamebocyte lysate preparation having reduced Factor G activity, themethod comprising the steps of: (a) admixing crude amebocyte lysate witha zwitterionic surfactant in an amount sufficient to produce a solutioncontaining a precipitate; and (b) removing said precipitate from saidsolution thereby to produce an amebocyte lysate preparation which isless reactive with (1→3)-β-D glucan than said crude amebocyte lysate.12. The method of claim 11, wherein said amebocyte lysate preparationreacts with a bacterial endotoxin.
 13. The method of claim 11, whereinsaid precipitate contains Factor G.
 14. The method of claim 11, whereinsaid zwitterionic surfactant is a sulfobetaine-type surfactant.
 15. Themethod of claim 14, wherein said sulfobetaine-type surfactant isselected from the group consisting of n-octyl- N,N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
 16. The method of claim 14,wherein said sulfobetaine surfactant is n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.
 17. The method of claim 11,comprising the additional step of removing said surfactant from saidsolution.
 18. The method of claim 17, wherein said surfactant is removedby organic solvent extraction.
 19. The method of claim 18, wherein saidorganic solvent is chloroform.
 20. A method of producing an improvedamebocyte lysate having reduced Factor G activity, the method comprisingthe steps of: (a) admixing crude amebocyte lysate with a n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate surfactant in an amountsufficient to produce a solution containing a precipitate; (b)separating said precipitate from said solution; and (c) removing saidsurfactant from said solution thereby to produce an amebocyte lysatepreparation that is reactive with bacterial endotoxin and is lessreactive with (1→3)-β-D glucan than said crude amebocyte lysate.
 21. Themethod of claim 20, wherein said surfactant is removed by organicsolvent extraction.
 22. The method of claim 21, wherein said organicsolvent is chloroform.
 23. The method of claim 2, 12, or 20 comprisingthe additional step of adding exogenous (1→3)-β-D glucan to saidamebocyte lysate preparation in an amount sufficient to enhance thesensitivity of said amebocyte lysate preparation to said endotoxinrelative to said amebocyte lysate preparation without said exogenous(1→3)-β-D glucan.
 24. The method of claim 23, wherein said exogenous(1→3)-β-D glucan is selected from the group consisting of LAL-RM,curdlan, pachyman, scleratan, leutinan, schizophyllan, coriolan,laminaran, and laminarin.
 25. The method of claim 23, wherein said(1→3)-β-D glucan is laminarin.
 26. An amebocyte lysate preparationproduced by the method of claim 1, 11, or
 20. 27. An amebocyte lysatepreparation produced by the method of claim
 23. 28. A compositioncomprising an amebocyte lysate preparation having reduced Factor Gactivity and exogenously added (1→3)-β-D glucan in an amount sufficientto enhance the sensitivity of said amebocyte lysate preparation toendotoxin relative to said amebocyte lysate preparation without saidexogenously added (1→3)-β-D glucan.
 29. The composition of claim 28,wherein said (1→3)-β-D glucan is selected from the group consisting ofcotton extract, cellulose acetate rinse, curdlan, pachyman, scleratan,leutinan, schizophyllan, coriolan, laminaran, and laminarin.
 30. Thecomposition of claim 28, wherein said (1→3)-β-D glucan is laminarin. 31.In a method of detecting a bacterial endotoxin in a sample, theimprovement comprising using the amebocyte lysate preparation of claim26.
 32. In a method of detecting a bacterial endotoxin in a sample, theimprovement comprising using the amebocyte lysate preparation of claim27.
 33. In a method of detecting a bacterial endotoxin in a sample, theimprovement comprising using the composition of claim 28 or 30.